Fluorescence Microscopy

Fluorescence Microscopy

Discovery

  1. British scientist Sir George G.Stokes first described fluorescence in 1852.
  2. He observed that the minimal  fluorspar emitted red light when it was illuminated by ultraviolet excitation.
  3. Stokes noted that fluorescence emission always occurred at a longer wavelength than of the excitation light.
  4. This shift towards longer wavelength is known as Stokes shift. 
Fluorescence Microscopy

Introduction to Fluorescence Microscopy

  1. Fluorescence microscopy is an one of the light microscope.
  2. It refers to any microscope that users fluorescence to generate an image.
  3. It produces 3D image.
  4. The technique is used to study specimen which can be made to fluorescence.

How does Fluorescence occurs

  1. Fluorescence is a phenomenon that takes place when a substances absorbs light at a given wavelength and emits light at another wavelength.
  2. Florescence occurs as an electron which has been excited to a higher and more unstable energy state, relaxes to its ground state and gives off a photon of light. 

Working Principle

  1. The sample to be analyzed is placed on a lens and the sample is coated with a fluorescence materials.
  2. The light is illuminated through the lens with the higher energy source. The illumination light is absorbed by the fluorophores.
  3. The sample causes them to emit a longer lower energy wavelength light.
  4. This fluorescence light can be separated from the surrounding radiation with filters. 
Fluorescence Microscopy

Dichoric Filter

  1. A dichoric filter or thin film is a very accurate color filter used to selective pass light of a small range of colors while reflecting other colors.
  2. The light from the light source is passed through the excitation.
  3. The specific wavelength of light is passed through the sample via dichromatic filter.
  4. The objective lens focuses the light to the specimen.
  5. The light emitted from the specimen is filtered by barrier filter.

Applications

  1. Imaging structural components of small specimens such as cells.
  2. Conducting viability studies on cell populations (are they alive or dead).
  3. Imaging the genetic material with a cell (DNA and RNA).
  4. To differentiate differentiate different types of cells. 

Fluorescence Microscopy

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